The experiments proposed here will analyze the folding of nascent proteins in an increasingly more complex and biologically relevant context. We will analyze how the folding of purified polypeptide fragments of increasing length, mimicking nascent polypeptides, differs from that of the corresponding full length proteins. Then, we will characterize the folding of individual nascent polypeptides in the context of translating ribosomes. This will allow us to assess how the folding energy landscape is influenced by the vectorial nature of protein synthesis and the unique environment of the ribosome. Finally, we will investigate how ribosome-associated chaperones modulate protein folding of both free and ribosome-bound nascent chains. These findings will allow us to map the folding energy landscapes of nascent proteins and provide insight into the fundamental principles that govern de novo protein folding. I have had a strong interest in the mechanism of de novo protein folding and its modulation by molecular chaperones since the beginning of my doctoral studies. Particularly, I have developed novel fluorescence-based methodologies to investigate de novo protein folding in the context of translation by the ribosome. In recent years, single molecule methodologies have become invaluable tools for the characterization of intrinsically heterogeneous protein folding pathways. Many of the applications that utilize optical tweezers to analyze biological processes with single molecule force spectroscopy have been developed in the laboratory of Prof. Bustamante. Thus, his laboratory provides an unequaled environment for me to become proficient in the utilization of optical tweezers and their application to protein folding. A postdoctorate in Prof. Bustamante's laboratory will enable me to build and operate optical tweezers and to study de novo protein folding with single molecule methods as an independent researcher. Ultimately, I would like to establish my own laboratory to engage PhD students and postdoctoral trainees in this fascinating area of research. RELEVANCE: Prevalent human disorders, such as Parkinson's and Alzheimer's diseases, are associated with protein misfolding. A more thorough understanding of protein folding and the function of molecular chaperones is imperative to gain insight into the pathophysiology underlying these disease states and may ultimately allow the development of effective therapeutic intervention.